Stable dispersion of solid particles comprising a water-insoluble pyrazine compound

ABSTRACT

A process for the preparation of a stable dispersion of solid particles, in an aqueous medium comprising combining (a) a first solution comprising a substantially water-insoluble substance which is a pyrazine compound of Formula I, a water-miscible organic solvent and an inhibitor with (b) an aqueous phase comprising water and optionally a stabilizer, thereby precipitating solid particles comprising the inhibitor and the substantially water-insoluble substance; and optionally removing the water-miscible organic solvent; wherein the inhibitor is a non-polymeric hydrophobic organic compound as defined in the description. 
     
       
         
         
             
             
         
       
     
     Also claimed are stable dispersions prepared by the process, solid particles prepared by the process and use of such particles.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application PCT/GB2004/000416, filed Feb. 2, 2004, whichclaims priority from United Kingdom Patent Application No. 0302673.9,filed Feb. 6, 2003, the specifications of each of which are incorporatedby reference herein. International Application PCT/GB2004/000416 waspublished under PCT Article 21(2) in English.

The present invention relates to a process for the preparation of astable dispersion of particles, particularly sub-micron particles in anaqueous medium and to a stable dispersion of particles in a liquidmedium, more particularly to a process for the preparation of adispersion of particles comprising a substantially water-insolublepharmacologically active pyrazine carboxamide compound of formula I inan aqueous medium, which particles exhibit substantially no increase insize upon storage in the aqueous medium, in particular to aqueousdispersions of particles that exhibit substantially no particle growthmediated by Ostwald ripening.

Dispersions of a solid material in a liquid medium are required for anumber of different applications including paints, inks, dispersions ofpesticides and other agrochemicals, dispersions of biocides anddispersions of pharmacologically active compounds. In the pharmaceuticalfield many pharmacologically active compounds have very low aqueoussolubility which can result in low bioavailability when such compoundsare administered to a patient. The bioavailability of such compounds maybe improved by reducing the particle size of the compound, particularlyto a sub-micron size, because this improves dissolution rate and henceabsorption of the compound.

The formulation of a pharmacologically active compound as an aqueoussuspension, particularly a suspension with a sub-micron particle size,enables the compound to be administered intravenously thereby providingan alternative route of administration which may increasebioavailability compared to oral administration.

Generally however, if there is a range of particles sizes dispersed in amedium there will be a differential rate of dissolution of the particlesin the medium. The differential dissolution results in the smallerparticles being thermodynamically unstable relative to the largerparticles and gives rise to a flux of material from the smallerparticles to the larger particles. The effect of this is that thesmaller particles dissolve in the medium, whilst material is depositedonto the larger particles thereby giving an increase in particle size.One such mechanism for particle growth is known as Ostwald ripening(Ostwald, Z Phys. Chem. (34), 1900, 495-503).

The growth of particles in a dispersion can result in instability of thedispersion during storage resulting in the sedimentation of particlesfrom the dispersion. It is particularly important that the particle sizein a dispersion of a pharmacologically active compound remains constantbecause a change in particle size is likely to affect thebioavailability and hence the efficacy of the compound. Furthermore, ifthe dispersion is required for intravenous administration, growth of theparticles in the dispersion may render the dispersion unsuitable forthis purpose, possibly leading to adverse or dangerous side effects.

Theoretically particle growth resulting from Ostwald ripening would beeliminated if all the particles in the dispersion were the same size.However, in practice, it is not possible to achieve a completely uniformparticle size and even small differences in particle sizes can give riseto particle growth.

Aqueous suspensions of a solid material can be prepared by mechanicalfragmentation, for example by milling. U.S. Pat. No. 5,145,648 describeswet milling of a suspension of a sparingly soluble compound in anaqueous medium. However, mechanical fragmentation of a material, forexample by milling, generally gives a wide distribution of particlesizes. Furthermore, mechanical fragmentation is less efficient in termsof particle size reduction when applied to non-crystalline startingmaterial.

U.S. Pat. No. 4,826,689 describes a processes for the preparation ofuniform sized particles of a solid by infusing an aqueous precipitatingliquid into a solution of the solid in an organic liquid undercontrolled conditions of temperature and infusion rate, therebycontrolling the particle size. U.S. Pat. No. 4,997,454 describes asimilar process in which the precipitating liquid is non-aqueous.However, when the particles have a small but finite solubility in theprecipitating medium particle size growth is observed after theparticles have been precipitated. To maintain a particular particle sizeusing these processes it is necessary to isolate the particles as soonas they have been precipitated to minimise particle growth. Therefore,particles prepared according to these processes cannot be stored in aliquid medium as a dispersion. Furthermore, for some materials the rateof Ostwald ripening is so great that it is not practical to isolatesmall particles (especially nano-particles) from the suspension.

W. J. Higuchi and J. Misra (J. Pharm. Sci., 51 (1962) 459) describe amethod for inhibiting the growth of the oil droplets in oil-in-wateremulsions by adding a hydrophobic compound (such as hexadecane) to theoil phase of the emulsion. U.S. Pat. No. 6,074,986 (WO95/07614)describes the addition of a polymeric material having a molecular weightof up to 10,000 to the disperse oil phase of an oil-in-water emulsion toinhibit Ostwald ripening. Welin-Berger et al. (Int. Jour. ofPharmaceutics 200 (2000) pp 249-260) describe the addition of ahydrophobic material to the oil phase of an oil-in-water emulsion toinhibit Ostwald ripening of the oil droplets in the emulsion. In theselatter three references the material added to the oil phase is dissolvedin the oil phase to give a single phase oil dispersed in the aqueouscontinuous medium.

EP 589 838 describes the addition of a polymeric stabilizer to stabilizean oil-in-water emulsion wherein the disperse phase is a hydrophobicpesticide dissolved in a hydrophobic solvent.

U.S. Pat. No. 4,348,385 discloses a dispersion of a solid pesticide inan organic solvent to which is added an ionic dispersant to controlOstwald ripening.

WO 99/04766 describes a process for preparing vesicular nano-capsules byforming an oil-in-water emulsion wherein the dispersed oil phasecomprises a material designed to form a nano-capsule envelope, anorganic solvent and optionally an active ingredient. After formation ofa stable emulsion the solvent is extracted to leave a dispersion ofnano-capsules.

U.S. Pat. No. 5,100,591 describes a process in which particlescomprising a complex between a water insoluble substance and aphospholipid are prepared by co-precipitation of the substance andphospholipid into an aqueous medium. Generally the molar ratio ofphospholipid to substance is 1:1 to ensure that a complex is formed.

U.S. Pat. No. 4,610,868 describes lipid matrix carriers in whichparticles of a substance is dispersed in a lipid matrix. The major phaseof the lipid matrix carrier comprises a hydrophobic lipid material suchas a phospholipid.

Patent Cooperation Treaty Application no PCT/GB02/05472 disclosespyrazine carboxamides of Formula I

and pharmaceutically acceptable salts, prodrugs, solvates andcrystalline forms thereof, in which

-   -   R¹ and R² independently represent:    -   a C₁₋₆alkyl group;    -   an (amino)C₁₋₄alkyl-group in which the amino is optionally        substituted by one or more C₁₋₃alkyl groups;        an optionally substituted non-aromatic C₃₋₁₅carbocyclic group;    -   a (C₃₋₁₂cycloalkyl)C₁₋₃alkyl-group;    -   a group —CH₂)_(r)(phenyl)_(s) in which r is 0, 1, 2, 3 or 4, s        is 1 when r is 0 otherwise s is 1 or 2 and the phenyl groups are        optionally independently substituted by one, two or three groups        represented by Z;    -   naphthyl;    -   anthracenyl;        a saturated 5 to 8 membered heterocyclic group containing one        nitrogen and optionally one of the following: oxygen, sulphur or        an additional nitrogen wherein the heterocyclic group is        optionally substituted by one or more C₁₋₃alkyl groups, hydroxy        or benzyl; 1-adamantylmethyl;        a group —(CH₂)_(t) Het in which t is 0, 1, 2, 3 or 4, and the        alkylene chain is optionally substituted by one or more        C₁₋₃alkyl groups and Het represents an aromatic heterocycle        optionally substituted by one, two or three groups selected from        a C₁₋₅alkyl group, a C₁₋₅alkoxy group or halo;        or R¹ represents H and R² is as defined above;        or R¹ and R² together with the nitrogen atom to which they are        attached represent a saturated 5 to 8 membered heterocyclic        group containing one nitrogen and optionally one of the        following: oxygen, sulphur or an additional nitrogen; wherein        the heterocyclic group is optionally substituted by one or more        C₁₋₃alkyl groups, hydroxy or benzyl;    -   X is CO or SO₂;    -   Y is absent or represents NH optionally substitututed by a        C₁₋₃alkyl group;    -   R³ and R⁴ independently represent phenyl, thienyl or pyridyl        each of which is optionally substituted by one, two or three        groups represented by Z;    -   Z represents a C₁₋₃alkyl group, a C₁₋₃alkoxy group, hydroxy,        halo, trifluoromethyl, trifluoromethylthio, trifluoromethoxy,        trifluoromethylsulphonyl, nitro, amino, mono or di        C₁₋₃alkylamino, mono or di C₁₋₃alkylamido, C₁₋₃alkylsulphonyl,        C₁₋₃alkoxycarbonyl, carboxy, cyano, carbamoyl, mono or di        C₁₋₃alkyl carbamoyl, sulphamoyl and acetyl; and    -   R⁵ is H, a C₁₋₃alkyl group, a C₁₋₃alkoxymethyl group,        trifluoromethyl, a hydroxyC₁₋₃alkyl group, C₁₋₃alkoxycarbonyl,        carboxy, cyano, carbamoyl, mono or di C₁₋₃alkylcarbamoyl,        acetyl, or hydrazinocarbonyl of formula —CONHNR^(a)R^(b) wherein        R^(a) and R^(b) are as previously defined for R¹ and R²        respectively;        and their use in the treatment of obesity, psychiatric and        neurological disorders. Such compounds are herineafter referred        to as a compound of Formula I.

We have surprisingly found that stable dispersions of solid particles ofa compound of Formula I in an aqueous medium can be prepared using aprecipitation process without the need for water-immiscible solvents orthe formation of an emulsion. The dispersions prepared according to thepresent invention exhibit little or no particle growth afterprecipitation mediated by Ostwald ripening.

According to a first aspect of the present invention there is provided aprocess for the preparation of a stable dispersion of solid particles inan aqueous medium comprising:

-   -   combining (a) a first solution comprising a substantially        water-insoluble substance which is a compound of Formula I, a        water-miscible organic solvent and an inhibitor with (b) an        aqueous phase comprising water and optionally a stabiliser,        thereby precipitating solid particles comprising the inhibitor        and the substantially water-insoluble substance; and optionally        removing the water-miscible organic solvent; wherein:        -   (i) the inhibitor is a non-polymeric hydrophobic organic            compound that is substantially insoluble in water;        -   (ii) the inhibitor is less soluble in water than the            substantially water-insoluble substance; and        -   (iii) the inhibitor is not a phospholipid.

The process according to the present invention enables stabledispersions of very small articles, especially nano-particles, to beprepared in high concentration without the need to quickly isolate theparticles from the liquid medium into which they have been precipitatedto prevent particle growth.

The dispersion according to the present invention is stable, by which wemean that the solid particles in the dispersion exhibit reduced orsubstantially no particle growth mediated by Ostwald ripening. By theterm “reduced particle growth” is meant that the rate of particle growthmediated by Ostwald ripening is reduced compared to particles preparedwithout the use of an inhibitor. By the term “substantialy no particlegrowth” is meant that the mean particle size of the particles in theaqueous medium does not increase by more than 10% (more preferably bynot more than 5%) over a period of 1 hour at 20° C. after precipitationinto the aqueous phase in the present process. Preferably the particlesexhibit substantially no particle growth.

It is to be understood that in those cases where the solid particles areprecipitated in an amorphous form the resulting particles will,generally, eventually revert to a thermodynamically more stablecrystalline form upon storage as an aqueous dispersion. The time takenfor such dispersions to re-crystallise is dependent upon the substanceand may vary from a few hours to a number of days. Generally suchre-crystallisation will result in particle growth and the formation oflarge crystalline particles which are prone to sedimentation from thedispersion. It is to be understood that the present invention does notprevent conversion of amorphous particles in the suspension into acrystalline state. The presence of the inhibitor in the particlesaccording to the present invention significantly reduces or eliminatesparticle growth mediated by Ostwald ripening, as hereinbefore described.The particles are therefore stable to Ostwald ripening and the term“stable” used herein is to be construed accordingly.

The solid particles in the dispersion preferably have a mean particlesize of less than 10 μm, more preferably less than 5 μm, still morepreferably less than 1 μm and especially less than 500 nm. It isespecially preferred that the particles in the dispersion have a meanparticle size of from 10 to 500 nm, more especially from 50 to 300 nmand still more especially from 100 to 200 nm. The mean size of theparticles in the dispersion may be measured using conventionaltechniques, for example by dynamic light scattering to measure theintensity-averaged particle size.

Generally the solid particles in the dispersion prepared according tothe present invention exhibit a narrow unimodal particle sizedistribution.

The solid particles may be crystalline, semi-crystalline or amorphous.In an embodiment, the solid particles comprise a compound of Formula Iin a substantially amorphous form. This can be advantageous as manypharmacological compounds exhibit increased bioavailability in amorphousform compared to their crystalline or semi-crystalline forms. Theprecise form of the particles obtained will depend upon the conditionsused during the precipitation step of the process. Generally, thepresent process results in rapid precipitation of the substance and theformation of substantially amorphous particles.

By substantially insoluble is meant a substance that has a solubility inwater at 25° C. of less than 0.5 mg/ml, preferably less than 0.1 mg/mland especially less than 0.05 mg/ml.

The greatest effect on particle growth inhibition is observed when thesubstance has a solubility in water at 25° C. of more than 0.05 μg/ml.In a preferred embodiment the substance has a solubility in the range offrom 0.05 μg/ml to 0.5 mg/ml, for example from 0.05 μg/ml to 0.05 mg/ml.

The solubility of the substance in water may be measured using aconventional technique. For example, a saturated solution of thesubstance is prepared by adding an excess amount of the substance towater at 25° C. and allowing the solution to equilibrate for 48 hours.Excess solids are removed by centrifugation or filtration and theconcentration of the substance in water is determined by a suitableanalytical technique such as HPLC.

Compound of Formula I

Further values of R¹, R², R³, R⁴ and R⁵ in compounds of Formula I nowfollow. It will be understood that such values may be used whereappropriate with any of the definitions, claims or embodiments definedhereinbefore or hereinafter.

In one group of compounds of Formula I, R¹ represents H, R² representscyclohexyl, X is CO and Y is absent.

In a second group of compounds of Formula I, R¹ and R² together with thenitrogen atom to which they are attached represent 1-piperidinyl.

In a third group of compounds of Formula I, R¹ represents H and R²represents phenyl.

A fourth group of compounds of Formula I is represented by formula Ia

and pharmaceutically acceptable salts, solvates and crystalline formsthereof, in which

-   -   R² represents cyclohexyl, 1-piperidinyl or phenyl;    -   R⁶ represents H, chloro, bromo, methyl or methoxy; and    -   when R⁷ represents H, R⁸ represents H or chloro; and    -   when R⁷ represents chloro, R⁵ represents H or chloro.

In a fifth group of compounds of formula I R⁵ is H.

In a sixth group of compounds of formula I X is CO.

In a seventh group of compounds of formula I X is SO₂.

In an eighth group of compounds of formula I Y is absent.

“Pharmaceutically acceptable salt”, where such salts are possible,includes both pharmaceutically acceptable acid and base addition salts.A suitable pharmaceutically acceptable salt of a compound of Formula Iis, for example, an acid-addition salt of a compound of Formula I whichis sufficiently basic, for example an acid-addition salt with aninorganic or organic acid such as hydrochloric, hydrobromic, sulphuric,trifluoroacetic, citric or maleic acid; or, for example a salt of acompound of Formula I which is sufficiently acidic, for example analkali or alkaline earth metal salt such as a sodium, calcium ormagnesium salt, or an ammonium salt, or a salt with an organic base suchas methylamine, dimethylamine, trimethylamine, piperidine, morpholine ortris-(2-hydroxyethyl)amine.

Throughout the specification and the appended claims, a given chemicalformula or name shall encompass all stereo and optical isomers andracemates thereof as well as mixtures in different proportions of theseparate enantiomers, where such isomers and enantiomers exist, as wellas pharmaceutically acceptable salts thereof and solvates thereof suchas for instance hydrates. Isomers may be separated using conventionaltechniques, e.g. chromatography or fractional crystallisation. Theenantiomers may be isolated by separation of racemate for example byfractional crystallisation, resolution or HPLC. The diastereomers may beisolated by separation of isomer mixtures for instance by fractionalcrystallisation, HPLC or flash chromatography. Alternatively thestereoisomers may be made by chiral synthesis from chiral startingmaterials under conditions which will not cause racemisation orepimerisation, or by derivatisation, with a chiral reagent. Allstereoisomers are included within the scope of the invention. Alltautomers, where possible, are included within the scope of theinvention.

The following definitions shall apply throughout the specification andthe appended claims.

Unless otherwise stated or indicated, the term “alkoxy” denotes either astraight or branched alkyl group. Examples of said alkyl include methyl,ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl and t-butyl.Preferred alkyl groups are methyl, ethyl, propyl, isopropyl and tertiarybutyl.

Unless otherwise stated or indicated, the term “alkoxy” denotes a groupO-alkyl, wherein alkyl is as defined above.

Unless otherwise stated or indicated, the term “halogen” shall meanfluorine, chlorine, bromine or iodine.

Specific compounds of the invention are one or more of the following:

-   N-(1-piperidinyl)-5,6-diphenyl-2-pyrazinecarboxamide;-   N-(1-piperidinyl)-5,6-bis(4-bromophenyl)-2-pyrazinecarboxamide;-   N-(1-piperidinyl)-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide;-   N-(1-piperidinyl)-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide;-   N-(1-piperidinyl)-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide;-   N-(1-piperidinyl)-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide;-   N-cyclohexyl-5,6-diphenyl-2-pyrazinecarboxamide;-   N-cyclohexyl-5,6-bis(4-bromophenyl)-2-pyrazinecarboxamide;-   N-cyclohexyl-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide;-   N-cyclohexyl-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide;-   N-cyclohexyl-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide;-   N-cyclohexyl-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide;-   N,5,6-triphenyl-2-pyrazinecarboxamide;-   N-phenyl-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide;-   N-phenyl-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide;-   N-phenyl-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide;-   N-phenyl-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide;-   N-(1-piperidinyl)-5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)-2-pyrazinecarboxamide;    and-   N-(1-piperidinyl)-6-(4-chlorophenyl)-5-(2,4-dichlorophenyl)-2-pyrazinecarboxamide;    and where applicable, optical isomers, tautomers, stereoisomers and    racemates thereof as well as pharmaceutically acceptable salts,    solvates and crystalline forms thereof.    Methods of Preparation

The compounds of Formula I may be prepared as outlined below accordingto any of the following methods. The compounds may also be prepared asdescribed for structurally related compounds in the prior art.

Compounds of formula I in which X is CO may be prepared by reacting acompound of formula II

in which R³, R⁴ and R⁵ are as previously defined with an amine offormula IIIR¹R² YNH₂  IIIin an inert solvent, for example dichloromethane, in the presence of acoupling agent, for example a carbodimide, eg1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and optionally in thepresence of a catalyst, for example a basic catalyst, eg4-dimethylamino-pyridine, at a temperature in the range of −25° C. to150° C.

Compounds of formula I in which X is SO₂ may be prepared by reacting acompound of formula IV

in which R³, R⁴ and R⁵ are as previously defined and A represents halowith an amine of formula IVR¹R² YNH₂  Vin an inert solvent, for example dichloromethane, and optionally in thepresence of a catalyst, for example a basic catalyst, eg4-dimethylamino-pyridine, at a temperature in the range of −25° C. to150° C.

Compounds of formulae I, III, IV and V may be prepared as described inthe Examples and by other methods known to those skilled in the art.Certain compounds of formulae II, III, IV and V are novel and areclaimed as a further aspect of the present invention as usefulintermediates. Specifically claimed are compounds of formula II in whichR³, R⁴ and R⁵ are as previously defined with the exception of5,6-diphenyl-2-pyrazinecarboxylic acid and5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxylic acid.

The compounds of the invention may be isolated from their reactionmixtures using conventional techniques.

Persons skilled in the art will appreciate that, in order to obtaincompounds of the invention in an alternative and in some occasions, moreconvenient manner, the individual process steps mentioned hereinbeforemay be performed in different order, and/or the individual reactions maybe performed at different stage in the overall route (i.e. chemicaltransformations may be performed upon different intermediates to thoseassociated hereinbefore with a particular reaction).

In the section above the expression “inert solvent” refers to a solventwhich does not react with the starting materials, reagents,intermediates or products in a manner which adversely affects the yieldof the desired product.

Inhibitor

The inhibitor is a non-polymeric hydrophobic organic compound that isless soluble in water than the substantially water-insoluble substancepresent in the first solution. Suitable inhibitors have a watersolubility at 25° C. of less than 0.1 mg/l, more preferably less than0.01 mg/l. In an embodiment of the invention the inhibitor has asolubility in water at 25° C. of less than 0.05 μg/ml, for example from0.1 ng/ml to 0.05 μg/ml.

In an embodiment of the invention the inhibitor has a molecular weightof less than 2000, such as less than 500, for example less than 400. Inanother embodiment of the invention the inhibitor has a molecular weightof less than 1000, for example less than 600. For example, the inhibitormay have a molecular weight in the range of from 200 to 2000, preferablya molecular weight in the range of from 400 to 1000, more preferablyfrom 400 to 600.

Suitable inhibitors include an inhibitor selected from classes (i) to(v) or a combination of two or more such inhibitors (for example amixture of an inhibitor and a co-inhibitor):

-   -   (i) a mono-, di- or (more preferably) a tri-glyceride of a fatty        acid. Suitable fatty acids include medium chain fatty acids        containing from 8 to 12, more preferably from 8 to 10 carbon        atoms or long chain fatty acids containing more than 12 carbon        atoms, for example from 14 to 20 carbon atoms, more preferably        from 14 to 18 carbon atoms. The fatty acid may be saturated,        unsaturated or a mixture of saturated and unsaturated acids. The        fatty acid may optionally contain one or more hydroxyl groups,        for example ricinoleic acid. The glyceride may be prepared by        well known techniques, for example, esterifying glycerol with        one or more long or medium chain fatty acids. In a preferred        embodiment the inhibitor is a mixture of triglycerides        obtainable by esterifying glycerol with a mixture of long or,        preferably, medium chain fatty acids. Mixtures of fatty acids        may be obtained by extraction from natural products, for example        from a natural oil such as palm oil. Fatty acids extracted from        palm oil contain approximately 50 to 80% by weight decanoic acid        and from 20 to 50% by weight of octanoic acid. The use of a        mixture of fatty acids to esterify glycerol gives a mixture of        glycerides containing a mixture of different acyl chain lengths.        Long and medium chain triglycerides are commercially available.        For example a preferred medium chain triglyceride (MCT)        containing acyl groups with 8 to 12, more preferably 8 to 10        carbon atoms is prepared by esterification of glycerol with        fatty acids extracted from palm oil, giving a mixture of        triglycerides containing acyl groups with 8 to 12, more        preferably 8 to 10 carbon atoms. This MCT is commercially        available as Miglyol 812N (Huls, Germany). Other commercially        available MCT's include Miglyol 810 and Miglyol 818 (Huls,        Germany). A further suitable medium chain triglyceride is        trilaurine (glycerol trilaurate). Commercially available long        chain trigylcerides include soya bean oil, sesame oil, sunflower        oil, castor oil or rape-seed oil.

Mono and di-glycerides may be obtained by partial esterification ofglycerol with a suitable fatty acid, or mixture of fatty acids. Ifnecessary the mono- and di-glycerides may be separated and purifiedusing conventional techniques, for example by extraction from a reactionmixture following esterification. When a mono-glyceride is used it ispreferably a long-chain mono glyceride, for example a mono glycerideformed by esterification of glycerol with a fatty acid containing 18carbon atoms;

-   -   (ii) a fatty acid mono- or (preferably) di-ester of a C₂₋₁₀        diol. Preferably the diol is an aliphatic diol which may be        saturated or unsaturated, for example a C₂₋₁₀-alkane diol which        may be a straight chain or branched chain diol. More preferably        the diol is a C₂₋₆-alkane diol which may be a straight chain or        branched chain, for example ethylene glycol or propylene glycol.        Suitable fatty acids include medium and long chain fatty acids        described above in relation to the glycerides. Preferred esters        are di-esters of propylene glycol with one or more fatty acids        containing from 8 to 10 carbon atoms, for example Miglyol 840        (Huls, Germany);    -   (iii) a fatty acid ester of an alkanol or a cycloalkanol.        Suitable alkanols include C₁₋₁₀-alkanols, more preferably        C₂₋₆-alcanols which may be straight chain or branched chain, for        example ethanol, propanol, isopropanol, n-butanol, sec-butanol        or tert-butanol. Suitable cycloalkanols include        C₃₋₆-cycloalkanols, for example cyclohexanol. Suitable fatty        acids include medium and long chain fatty acids described above        in relation to the glycerides. Preferred esters are esters of a        C₂₋₆-alkanol with one or more fatty acids containing from 8 to        10 carbon atoms, or more preferably 12 to 29 carbon atoms, which        fatty acid may saturated or unsaturated. Suitable esters        include, for example isopropyl myristrate or ethyl oleate;    -   (iv) a wax. Suitable waxes include esters of a long chain fatty        acid with an alcohol containing at least 12 carbon atoms. The        alcohol may an aliphatic alcohol, an aromatic alcohol, an        alcohol containing aliphatic and aromatic groups or a mixture of        two or more such alcohols. When the alcohol is an aliphatic        alcohol it may be saturated or unsaturated. The aliphatic        alcohol may be straight chain, branched chain or cyclic.        Suitable aliphatic alcohols include those containing more than        12 carbon atoms, preferably more than 14 carbon atoms especially        more than 18 carbon atoms, for example from 12 to 40, more        preferably 14 to 36 and especially from 18 to 34 carbon atoms.        Suitable long chain fatty acids include those described above in        relation to the glycerides, preferably those containing more        than 14 carbon atoms especially more than 18 carbon atoms, for        example from 14 to 40, more preferably 14 to 36 and especially        from 18 to 34 carbon atoms. The wax may be a natural wax, for        example bees wax, a wax derived from plant material, or a        synthetic wax prepared by esterification of a fatty acid and a        long chain alcohol. Other suitable waxes include petroleum waxes        such as a paraffin wax;    -   (v) a long chain aliphatic alcohol. Suitable alcohols include        those with 6 or more carbon atoms, more preferably 8 or more        carbon atoms, such as 12 or more carbon atoms, for example from        12 to 30, for example from 14 to 20 carbon atoms. It is        especially preferred that the long chain aliphatic alcohol has        from 6 to 20, more especially from 6 to 14 carbon atoms, for        example from 8 to 12 carbon atoms. The alcohol may be straight        chain, branched chain, saturated or unsaturated. Examples of        suitable long chain alcohols include, 1-hexanol, 1-decanol,        1-hexadecanol, 1-octadecanol, or 1-heptadecanol (more preferably        1-decanol); or    -   (vi) a hydrogenated vegetable oil, for example hydrogenated        castor oil.

In one embodiment of the present invention the inhibitor is selectedfrom a medium chain triglyceride and a long chain aliphatic alcoholcontaining from 6 to 12, preferably from 10 to 20 carbon atoms.Preferred medium chain triglycerides and long chain aliphatic alcoholsare as defined above. In a preferred embodiment the inhibitor isselected from a medium chain triglyceride containing acyl groups withfrom 8 to 12 carbon atoms or a mixture of such triglycerides (preferablyMiglyol 812N) and an aliphatic alcohol containing from 10 to 14 carbonatoms (preferably 1-decanol) or a mixture thereof (for example a mixturecomprising Miglyol 812N and 1-decanol).

Suitably the inhibitor is a liquid at the temperature at which thedispersion is prepared. Preferably the inhibitor is liquid at ambienttemperature (25° C.).

When the substantially water-insoluble substance is a pharmacologicallyactive compound the inhibitor is preferably a pharmaceutically inertmaterial.

The inhibitor is present in the particles in a quantity sufficient toprevent Ostwald ripening of the particles in the suspension. Preferablythe inhibitor will be the minor component in the solid particles formedin the present process comprising the inhibitor and the substantiallywater-insoluble substance. Preferably, therefore, the inhibitor ispresent in a quantity that is just sufficient to prevent Ostwaldripening of the particles in the dispersion, thereby minimising theamount of inhibitor present in the particles.

In embodiments of the present invention the weight fraction of inhibitorrelative to the total weight of inhibitor and substantiallywater-insoluble substance (i.e. weight of inhibitor/weight ofinhibitor+weight of substantially water-insoluble substance) is from0.01 to 0.99, preferably from 0.01 to 0.5, especially from 0.05 to 0.3and more especially from 0.06 to 0.25. In a preferred embodiment theweight fraction of inhibitor relative to the total weight of inhibitorand substantially water-insoluble substance is less than 0.5, morepreferably 0.3 or less, for example from 0.05 to 0.3, such as from 0.06to 0.25, for example about 0.2. This is particularly preferred when thesubstantially water-insoluble substance is a pharmacologically activesubstance because high levels of inhibitor (e.g. a weight fraction above0.5) may give rise to unwanted side effects and/or affect thedissolution rate/bioavalability of the pharmacologically activesubstance when administered in vivo.

Furthermore, we have found that in general a low weight ratio ofinhibitor to the inhibitor and the substantially water-insolublesubstance which is a compound of Formula I (i.e. less than 0.5) issufficient to prevent particle growth by Ostwald ripening, therebyallowing small (preferably less than 1 μm, preferably less than 500 nm)stable particles to be prepared. A small and constant particle size isoften desirable, especially when the substantially water-insolublesubstance is a pharmacologically active material that is used, forexample, for intravenous administration.

One application of the dispersions prepared by the process according tothe present invention is the study of the toxicology of compounds ofFormula I. The dispersions prepared according to the present process canexhibit improved bioavailability compared to dispersions prepared usingalternative processes, particularly when the particle size of thesubstance is less than 0.5 μm. In this application it is advantageous tominimise the amount of inhibitor relative to the active compound so thatany effects on the toxicology associated with the presence of theinhibitor are minimised.

When the substantially water-insoluble substance has an appreciablesolubility in the inhibitor the weight ratio of inhibitor tosubstantially water-insoluble substance should be selected to ensurethat the amount of substantially water-insoluble substance exceeds thatrequired to form a saturated solution of the substantiallywater-insoluble substance in the inhibitor. This ensures that solidparticles of the substantially water-insoluble substance are formed inthe dispersion. This is important when the inhibitor is a liquid at thetemperature at which the dispersion is prepared (for example ambienttemperature) to ensure that the process does not result in the formationliquid droplets comprising a solution of the substantiallywater-insoluble substance in the inhibitor, or a two phase systemcomprising the solid substance and large regions of the liquidinhibitor.

Without wishing to be bound by theory we believe that systems in whichthere is a phase separation between the substance and inhibitor in theparticles are more prone to Ostwald ripening than those in which thesolid particles form a substantially single phase system. Accordingly,in a preferred embodiment the inhibitor is sufficiently miscible in thesubstantially water-insoluble material to form solid particles in thedispersion comprising a substantially single-phase mixture of thesubstance and the inhibitor. The composition of the particles formedaccording to the present invention may be analysed using conventionaltechniques, for example analysis of the (thermodynamic) solubility ofthe substantially water-insoluble substance in the inhibitor, meltingentropy and melting points obtained using routine differential scanningcalorimetry (DSC) techniques to thereby detect phase separation in thesolid particles. Furthermore, studies of nano-suspensions using nuclearmagnetic resonance (NMR) (e.g. line broadening of either component inthe particles) may be used to detect phase separation in the particles.

Generally the inhibitor should have a sufficient miscibility with thesubstance to form a substantially single phase particle, by which ismeant that the inhibitor is molecularly dispersed in the solid particleor is present in small domains of inhibitor dispersed throughout thesolid particle. It is thought that for many substances thesubstance/inhibitor mixture is a non-ideal mixture by which is meantthat the mixing of two components is accompanied by a non-zero enthalpychange.

An indication of the substance/inhibitor miscibility in the solidparticles is provided by the interaction parameter χ for thesubstance-inhibitor mixture. The χ parameter may be derived from thewell known Bragg-Williams, Flory-Huggins or the Regular Solutiontheories (see e.g. Jönsson, B. Lindman, K. Holnberg, B. Kronberg,“Surfactants and Polymers in Solution”, John Wiley & Sons, 1998 and Neauet al, Pharmaceutical Research, 14, 601 1997). In an ideal mixture χ is0, and according to the Bragg-Williams theory a two-component mixturewill not phase separate provided χ<2. We believe that in many particlesprepared according to the present invention the substance and inhibitorare not ideal mixtures and therefore the χ value is not zero.

We have surprisingly found that when χ is <2.5 the solid particlesprepared according to the invention exhibit little or no Ostwaldripening. Those systems in which χ is >2.5 are thought to be prone tophase separation and are less stable to Ostwald ripening. Suitably the χvalue of the substance-inhibitor mixture is 2 or less, for example from0 to 2, preferably 0.1 to 2, such as 0.2 to 1.8.

Many small molecule organic substances (Mw<1000) are available in acrystalline form or can be prepared in crystalline form usingconventional techniques (for example by recrystallisation from asuitable solvent system). In such cases the χ parameter of the substanceand inhibitor mixture is easily determined from the Equation I:

$\chi = {\frac{{{- \Delta}\; S_{m}{\ln\left\lbrack {T_{m}/T} \right\rbrack}R} - {\ln\; x_{1}^{s}}}{\left( {1 - x_{1}^{s}} \right)^{2}}\mspace{295mu}{{Equation}\mspace{14mu} I}}$

-   -   wherein:    -   ΔS_(m) is the entropy of melting of the crystalline        substantially water-insoluble substance (measured using a        conventional technique such as DSC measurement);    -   T_(m) is the melting point (K) of the crystalline substantially        water-insoluble substance (measured using a conventional        technique such as DSC measurement);    -   T is the temperature of the dispersion (K);    -   R is the gas constant; and    -   x^(s) ₁ is the mole fraction solubility of the crystalline        substantially water-insoluble substance in the inhibitor        (measured using conventional techniques for determining        solubility for example as hereinbefore described). In the above        equation T_(m) and ΔS_(m) refer to the melting point of the        crystalline form of the material. In those cases where the        substance may exist in the form of different polymorphs, T_(m)        and ΔS_(m) are determined for the polymorphic form of the        substance that is most stable at the temperature of the        dispersion. As will be understood, the measurement of ΔS_(m),        and x^(s) ₁ are performed on the crystalline substantially        water-insoluble substance prior to formation of the dispersion        according to the invention and thereby enables a preferred        inhibitor for the substantially water-insoluble material to be        selected by performing simple measurements on the bulk        crystalline material.

The mole fraction solubility of the crystalline substantiallywater-insoluble substance in the inhibitor (x^(s) ₁) is simply thenumber of moles of substance per mole of inhibitor present in asaturated solution of the substance in the inhibitor. As will berealized the equation above is derived for a two component system of asubstance and an inhibitor. In those systems where the inhibitorcontains more than one compound (for example in the case of a mediumchain triglyceride comprising a mixture of triglycerides such as Miglyol812N, or where a mixture of inhibitors is used) it is sufficient tocalculate x^(s) ₁ in terms of the “apparent molarity” of the mixture ofinhibitors. The apparent molarity of such a mixture is calculated for amixture of n inhibitor components to be:

${{Apparent}\mspace{14mu}{molarity}} = \frac{{Mass}\mspace{14mu}{of}\mspace{14mu} 1\mspace{14mu}{litre}\mspace{14mu}{of}\mspace{14mu}{inhibitor}\mspace{14mu}{{mixture}.}}{\left\lbrack {\left( {a*{Mwa}} \right) + \left( {b*{Mwb}} \right) + {\ldots\mspace{11mu}\left( {n*{Mwn}} \right)}} \right\rbrack}$wherein: a, b . . . n are the weight fraction of each component in theinhibitor mixture (for example for component a this is % w/w componenta/100); andMwa . . . . Mwn is the molecular weight of each component a. n in themixture.

x^(s) ₁ is then calculated as:

$x_{1}^{s} = \frac{\begin{matrix}{{Molar}\mspace{14mu}{solubility}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{crystalline}\mspace{14mu}{substance}} \\{{in}\mspace{14mu}{the}\mspace{14mu}{inhibitor}\mspace{14mu}{mixture}\mspace{14mu}\left( {{mol}\text{/}l} \right)}\end{matrix}}{{Apparent}\mspace{14mu}{molarity}\mspace{14mu}{of}\mspace{14mu}{inhibitor}\mspace{14mu}{mixture}\mspace{14mu}\left( {{mol}\text{/}l} \right)}$

When the inhibitor is a solid at the temperature that the dispersion isprepared, the mole fraction solubility, x^(s) ₁, can be estimated bymeasuring the mole fraction solubility at a series of temperatures abovethe melting point of the inhibitor and extrapolating the solubility backto the desired temperature. However, as hereinbefore mentioned, it ispreferred that the inhibitor is a liquid at the temperature that thedispersion is prepared. This is advantageous because, amongst otherthings, the use of a liquid inhibitor enables the value of x^(s) ₁ to bemeasured directly.

In certain cases, it may not be possible to obtain the substantiallywater-insoluble material in a crystalline form. In such cases, preferredinhibitors are those which are sufficiently miscible with thesubstantially water-insoluble material to form a substantially singlephase mixture when mixed in the required substance:inhibitor ratio.Miscibility of the inhibitor in the substantially water-insolublematerial may be determined using routine experimentation. For examplethe substance and inhibitor may be dissolved in a suitable organicsolvent followed by removal of the solvent to leave a mixture of thesubstance and inhibitor. The resulting mixture may then be characterisedusing a routine technique such as DSC characterisation to determinewhether or not the mixture is a single phase system. This empiricalmethod enables preferred inhibitors for a particular substance to beselected and will provide substantially single phase solid particles inthe dispersion prepared according to the present invention.

In a further embodiment of the present invention the miscibility of thesubstance and the inhibitor may be increased by the addition of asuitable co-inhibitor to the first solution in the present process. Thepresence of the co-inhibitor increases the miscibility of the substanceand the inhibitor mixture, thereby reducing the χ value and furtherreducing or preventing Ostwald ripening. Suitable co-inhibitors includean inhibitor as hereinbefore defined, preferably an inhibitor selectedfrom classes (i) to (v) listed hereinbefore. In a preferred embodimentwhen the inhibitor is a medium chain triglyceride containing acyl groupswith 8 to 12 carbon atoms (or a mixture of such triglycerides such asMiglyol 812N), a preferred co-inhibitor is a long chain aliphaticalcohol containing 6 or more carbon atoms (preferably from 6 to 14carbon atoms) for example 1-hexanol or more preferably 1-decanol. Theweight ratio of inhibitor:co-inhibitor is selected to give the desired χvalue of the substance/inhibitor/co-inhibitor mixture and may be variedover wide limits, for example from 10:1 to 1:10, such as approximately1:1. Preferred values for χ are as hereinbefore defined.

The inhibitor in the present invention is not a phospholipid. Suchlipids have a hydrophilic phosphorous containing “head” groups and oneor more lipophilic “tail” groups. Such phosphlipids are capable offorming lipid bilayers and exhibit surface-active effects. Examples ofphospholipids excluded from the present invention include, for examplethe phospholipids described in U.S. Pat. No. 5,100,591.Water-Miscible Organic Solvent

The water-miscible organic solvent in the first phase is preferablymiscible with water in all proportions. The water-miscible organicsolvent should also be a solvent for both the substantiallywater-insoluble substance and the inhibitor. The water-miscible organicsolvent is selected such that the inhibitor and the substantiallywater-insoluble substance each have a sufficient solubility in the watermiscible organic solvent to enable a precipitate of the substantiallywater-insoluble substance to form when the first solution is combinedwith the aqueous phase. Suitably, the inhibitor and the substantiallywater-insoluble substance each have a solubility of 10 mg/ml or more inthe water-miscible organic solvent.

Generally it is preferred that the concentration of the substantiallywater-insoluble substance in the water-miscible organic solvent is ashigh as possible to aid efficient precipitation. The upper concentrationof the substantially water-insoluble substance in the water-miscibleorganic solvent is determined by the solubility of the substance in thesolvent.

However, we have found that a wide range of concentrations may be usedin the present process. Typically, a concentration of substantiallywater-insoluble substance of 1% by weight or more in the organic solventis sufficient.

The inhibitor and/or the substantially water-insoluble substance shouldbe completely dissolved in the water-miscible organic solvent. Thepresence of particles of the inhibitor and/or the substantiallywater-insoluble substance in the first solution may result in poorcontrol of the particle size distribution in the dispersion.

If required the solubility of the inhibitor and/or the substantiallywater-insoluble substance in the water-miscible organic solvent can beincreased by heating a mixture of the inhibitor, substantiallywater-insoluble substance and water-miscible organic solvent to providea solution. The solution is then maintained at elevated temperatureuntil it is combined with the aqueous phase in the process.

As will be understood, the selection of water-miscible organic solventwill be dependent upon the nature of the substantially water-insolublesubstance. When the substantially water-insoluble substance is anorganic compound the water-miscible organic solvent should have asufficiently low dielectric constant to be able to dissolve thesubstantially water-insoluble substance and the inhibitor. Suitablewater-miscible solvents for dissolving a substantially water-insolubleorganic substance include, a water-miscible alcohol, for examplemethanol, ethanol, n-propyl alcohol, isopropyl alcohol, tert-butylalcohol, ethylene glycol or propylene glycol; dimethylsulfoxide;dimethylformamide; a water-miscible ether, for example tetrahydrofuran;a water-miscible nitrile, for example acetonitrile; a water-miscibleketone, for example acetone or methyl ethyl ketone; an amide, forexample dimethylacetamide or a mixture of two or more of the abovementioned water-miscible organic solvents. A preferred water-miscibleorganic solvent is dimethylacetamide (DMA).

Precipitation

In the present process the first solution and the aqueous phase may becombined by adding the first solution to the aqueous phase.Alternatively, the aqueous phase may be added to the first solution.During the combination of the first solution and the aqueous phase theconditions are controlled to give precipitated solid particles of therequired particle size. The particle size resulting from the combinationof the first solution and aqueous phase is determined by a number offactors including, the rate of agitation during the combination of thefirst solution and the aqueous phase, the temperature during thecombination and the rate at which the combination takes place. As willbe clear, sufficient aqueous phase is used during the combination toextract sufficient water-miscible organic solvent from the firstsolution to cause precipitation of the solid particles from the firstsolution.

Suitable conditions for the addition of the aqueous phase to the firstsolution for the formation of sub-micron particles are described in U.S.Pat. No. 4,826,689, incorporated herein by reference thereto, wherein anaqueous phase is injected into an agitated phase containing thesubstance dissolved in an organic solvent. Suitable rates of additionare typically from 100 ml/min to 1000 ml/min per 50 ml of the firstsolution. A suitable temperature for the addition is from 0 to 100° C.,more preferably from 5 to 50° C.

Addition of the aqueous phase into the first solution may be achievedusing a number of techniques, for example by injecting the aqueous phasedirectly into the first solution (for example via a syringe) or byadding the aqueous phase drop-wise into the first solution. For largerscale production the aqueous phase may be added to the first solutionusing a flow mixer. Preferably the first solution is agitated duringaddition of the aqueous phase by for example stirring, preferably at arate sufficient to induce a high degree of turbulence in the firstsolution and hence a very rapid precipitation and distribution ofparticles into the liquid medium of the dispersion. Alternatively, thefirst solution may be agitated by sonication in an ultrasonic bath.

When the first solution is added to the aqueous phase, the aqueous phaseis preferably agitated as described above, thereby enhancing extractionof water-miscible solvent from the first solution to give smallparticles and good dispersion of the particles in the liquid medium.Suitable rates and methods of addition, temperature and degree ofagitation are analogous to those described above for the addition of theaqueous phase into the first solution.

Some particles will precipitate and form a uniform dispersion withoutthe need for a stabiliser in the aqueous phase. However, we have foundthat many particles tend to aggregate upon precipitation unless astabiliser is present in the aqueous phase.

Stabilisers suitable for the prevention of particle aggregation indispersions are well known to those skilled in the art. Suitablestabilisers include dispersants and surfactants (which may be anionic,cationic or non-ionic) or a combination thereof. Suitable dispersantsinclude, a polymeric dispersant, for example a polyvinylpyrrolidone, apolyvinylalcohol or a cellulose derivative, for examplehydroxypropylmethyl cellulose, hydroxy ethyl cellulose,ethylhydroxyethyl cellulose or carboxymethyl cellulose. Suitable anionicsurfactants include alkyl and aryl sulphonates, sulphates orcarboxylates, such as an alkali metal alkyl and aryl sulphonate orsulphate, for example, sodium dodecyl sulphate. Suitable cationicsurfactants include quaternary ammonium compounds and fatty amines.Suitable non-ionic surfactants include, monoesters of sorbitan which mayor may not contain a polyoxyethylene residue, ethers formed betweenfatty alcohols and polyoxyethylene glycols,polyoxyetheylene-polypropylene glycols, an ethoxylated castor oil (forexample Cremophor EL), ethoxylated hydrogenated castor oil, ethoxylated120H-stearic acid (for example Solutol HS15). The aqueous phase maycontain a single stabiliser or a mixture of two or more stabilisers. Ina preferred embodiment the aqueous phase contains a polymeric dispersantand a surfactant (preferably an anionic surfactant), for example apolyvinylpyrrolidone and sodium dodecyl sulphate. When the substantiallywater-insoluble material is a pharmacologically active compound it ispreferred that the stabiliser is a pharmaceutically acceptable material.

Generally the aqueous phase will contain from 0.01 to 1% by weight,preferably from 0.05 to 0.5% by weight and especially from 0.1 to 0.2%by weight of stabiliser. We have found that the dispersions preparedaccording to the present process require lower levels of stabilisers(such as surfactants) compared to precipitation processes that do notuse an inhibitor.

Optionally, additional stabiliser may be added to the dispersion afterprecipitation of the particles into the aqueous phase to provideadditional inhibition of particle aggregation in the dispersion.

The combination of the first solution and aqueous phase in the processaccording to the present invention results in very fast, substantiallyinstantaneous precipitation of particles of the inhibitor andsubstantially water-insoluble material to give particles of the desiredsize with a narrow particle size distribution. The precipitation avoidsthe need to form an emulsion prior to extraction of the water-miscibleorganic solvent, and thereby considerably simplifies the preparation ofa dispersion of solid particles compared to emulsion-based processes.

Optionally the water-miscible organic solvent can be removed from thedispersion after the precipitation. Suitable methods for removing thewater-miscible organic solvent include evaporation, for example byheating the dispersion under vacuum, reverse osmosis, dialysis,ultra-filtration or cross-flow filtration. The dispersion may beconcentrated after precipitating the particles by removing excess waterfrom the dispersion, for example by evaporation, spray drying orlyophilisation.

Optionally additional components may be added to the dispersion forexample viscosity modifying agents, buffers, taste masking agents,anti-oxidants, preservatives or colorants. The additional components maybe added before, or more preferably, after the precipitation of theparticles.

According to a further embodiment of the present invention there isprovided a process for the preparation of a stable dispersion of solidparticles of a substantially water-insoluble substance which is acompound of Formula I in an aqueous medium comprising:

-   -   combining (a) a first solution comprising the substantially        water-insoluble compound of Formula I, a water-miscible organic        solvent and an inhibitor with (b) an aqueous phase comprising        water and optionally a stabiliser, thereby precipitating solid        particles comprising the inhibitor and the substantially        water-insoluble pharmacologically active substance; and        optionally removing the water-miscible organic solvent;    -   wherein the inhibitor is less soluble in water than the        pharmacologically active substance, which inhibitor is selected        from one or more of:        -   (i) a mono-, di- or (more preferably) a tri-glyceride of a            fatty acid;        -   (ii) a fatty acid mono- or (preferably) di-ester of a C₂₋₁₀            diol;        -   (iii) a fatty acid ester of an alkanol or a cycloalkanol;        -   (iv) a wax;        -   (v) a long chain aliphatic alcohol (preferably containing 6            or more carbon atoms, for example from 8 to 12 carbon            atoms); and        -   (vi) a hydrogenated vegetable oil.

This embodiment of the present invention provides stable dispersions ofparticles of a solid substantially water-insoluble substance which is acompound of Formula I in an aqueous medium. The dispersions preparedaccording to this embodiment exhibit little or no growth in particlesize during storage (resulting from, Ostwald ripening).

In this embodiment it is preferred that the miscibility of thesubstantially water-insoluble substance and inhibitor are sufficient togive substantially single phase solid particles in the dispersion, morepreferably the inhibitor/substance mixture has a χ value of <2.5, morepreferably 2 or less, for example from 0 to 2, preferably from 0.1 to 2wherein the χ value is as hereinbefore defined.

In this embodiment the inhibitor is preferably a medium chaintri-glyceride (MCT) containing acyl groups with 8 to 12 (more preferably8 to 10) carbon atoms, or a mixture thereof, for example Miglyol 812N.The miscibility of the inhibitor with the substance may be increased byusing a co-inhibitor as hereinbefore described. For example, a suitableinhibitor/co-inhibitor in this embodiment comprises a medium chaintri-glyceride (MCT) as defined above and a long chain aliphatic alcoholhaving 6 to 12 (more preferably 8 to 12, for example 10) carbon atoms,or a mixture comprising two or more such inhibitors (for example1-hexanol or (more preferably) 1-decanol). A preferredinhibitor/co-inhibitor for use in this embodiment is a mixture ofMiglyol 812N and 1-decanol.

If required the particles present in the dispersion prepared accordingto the present invention may be isolated from the aqueous mediumfollowing precipitation (or removal of the water-miscible organicsolvent, if used). The particles may be separated using conventionaltechniques, for example by centrifuging, reverse osmosis, membranefiltration, lyophilisation or spray drying. Isolation of the particlesis useful when the particles comprise a substantially water-insolublepharmacologically active compound of Formula I because it allows theparticles to be washed and re-suspended in a sterile aqueous medium togive a suspension suitable for administration to a warm blooded mammal(especially a human), for example by oral or parenteral (e.g.intravenous) administration.

In this embodiment an agent may be added to the suspension prior toisolation of the particles to prevent agglomeration of the solidparticles during isolation (for example spray drying or lyophilisation).Suitable agents include for example a sugar such as mannitol. Isolationof the particles from the suspension is also useful when it is desirableto store the particles as a powder. The powder may then be re-suspendedin an aqueous medium prior to use. This is particularly useful when thesubstantially water-insoluble substance is a pharmacologically activecompound of Formula I. The isolated particles of the substance may thenbe stored as a powder in, for example a vial and subsequently bere-suspended in a suitable liquid medium for administration to a patientas described above.

Alternatively the isolated particles may be used to prepare solidformulations, for example by blending the particles with suitableexcipients/carriers and granulating or compressing the resulting mixtureto form a tablet or granules suitable for oral administration.Alternatively the particles may be suspended, dispersed or encapsulatedin a suitable matrix system, for example a biocompatible polymericmatrix, for example a hydroxypropyl methylcellulose (HPMC) orpolylactide/glycloide polymer to give a controlled or sustained releaseformulation.

In another embodiment of the present invention the process is performedunder aseptic conditions, thereby providing a sterile dispersiondirectly which can be administered to a warm blooded mammal as describedabove without the need for additional purification or sterilisationsteps. Alternatively, the dispersion may be sterile filtered followingprecipitation and optional removal of the water-miscible organic solventto leave a sterile suspension.

According to a further aspect of the present invention there is provideda stable aqueous dispersion comprising a continuous aqueous phase inwhich is dispersed solid particles comprising an inhibitor and asubstantially water-insoluble substance which is a compound of FormulaI, wherein said dispersion is obtainable by the process according to thepresent invention; and wherein:

-   -   (i) the inhibitor is a non-polymeric hydrophobic organic        compound that is substantially insoluble in water;    -   (ii) the inhibitor is less soluble in water than the        substantially water-insoluble substance; and    -   (iii) the inhibitor is not a phospholipid.

The dispersion according to this aspect of the present invention exhibitlittle or no particle growth upon storage, mediated by Ostwald ripening(i.e. the dispersion is a stable dispersion as defined above in relationto the first aspect of the invention).

The particles preferably have a mean diameter of less than 1 μm and morepreferably less than 500 nm. It is especially preferred that theparticles in the dispersion have a mean particle size of from 10 to 500nm, more especially from 50 to 300 nm and still more especially from 100to 200 nm.

The weight fraction of inhibitor in the particles is preferably lessthan 0.5, more preferably 0.3 or less, for example from 0.05 to 0.3,preferably from 0.06 to 0.25.

It is preferred that the miscibility of the substantiallywater-insoluble material and inhibitor are sufficient to givesubstantially single phase solid particles, more preferably theinhibitor/substance mixture has a χ value of <2.5, more preferably 2 orless, for example from 0 to 2, preferably from 0.1 to 2, wherein the χvalue is as hereinbefore defined.

The particles may contain a single compound of Formula I or two or moresuch substances. The particles may contain a single inhibitor or acombination of an inhibitor and one or more co-inhibitors ashereinbefore described.

The dispersions according to the present invention may be administeredto a warm blooded mammal (especially a human), for example by oral orparenteral (e.g. intravenous) administration. In an alternativeembodiment the dispersion may be used as a granulation liquid in a wetgranulation process to prepare granules comprising the substantiallywater-insoluble pharmacologically active material and one or moreexcipients (optionally after first concentrating the dispersion byremoval of excess aqueous medium). The resulting granules may then beused directly, for example by filling into capsules to provide a unitdosage containing the granules. Alternatively the granules may beoptionally mixed with further excipients, disintegrants, binders,lubricants etc. and compressed into a tablet suitable for oraladministration. If required the tablet may be coated to provide controlover the release properties of the tablet or to protect it againstdegradation, for example through exposure to light and/or moisture. Wetgranulation techniques and excipients suitable for use in tabletformulations are well known in the art.

According to a further aspect of the present invention there is provideda solid particle comprising an inhibitor and a substantiallywater-insoluble substance which is a compound of Formula I obtainable bythe process according to the present invention, wherein the substanceand the inhibitor are as hereinbefore defined in relation to the firstaspect of the present invention.

According to a further aspect of the present invention there is provideda solid particle comprising an inhibitor and a substantiallywater-insoluble substance which is a compound of Formula I obtainable bythe process according to the present invention, for use as a medicament,According to a further aspect of the present invention there is provideda pharmaceutical composition comprising a pharmaceutically acceptablecarrier or diluent in association with a solid particle comprising aninhibitor and a substantially water-insoluble pharmacologically activesubstance which is a compound of Formula I obtainable by the processaccording to the present invention.

Suitable pharmacutically acceptable carriers or diluents are well knownexcipients used in the preparation of pharmaceutical formulations, forexample, fillers, binders, lubricants, disintegrants and/or releasecontrolling/modifying excipients.

According to a further aspect of the present invention there is provideda method for inhibiting Ostwald ripening in a dispersion of solidsubstantially water-insoluble particles in an aqueous medium comprising:

combining (a) a first solution comprising a substantiallywater-insoluble substance which is a compound of Formula I, awater-miscible organic solvent and an inhibitor with (b) an aqueousphase comprising water and optionally a stabiliser, therebyprecipitating solid particles comprising the inhibitor and thesubstantially water-insoluble substance to give a dispersion of thesolid substantially water-insoluble particles in an aqueous medium; andoptionally removing the water-miscible organic solvent from thedispersion;

wherein:

-   -   (i) the inhibitor is a non-polymeric hydrophobic organic        compound that is substantially insoluble in water;    -   (ii) the inhibitor is less soluble in water than the        substantially water-insoluble substance; and    -   (iii) the inhibitor is not a phospholipid.

Preferred inhibitors and substantially water-insoluble substances foruse in this embodiment are as hereinbefore defined in relation to thefirst aspect of the present invention.

According to a further aspect of the present invention there is providedthe use of an inhibitor to prevent or inhibit Ostwald ripening in adispersion of solid substantially water-insoluble particles in anaqueous medium wherein:

-   -   (i) the inhibitor is a non-polymeric hydrophobic organic        compound that is substantially insoluble in water;    -   (ii) the inhibitor is less soluble in water than the        substantially water-insoluble substance; and    -   (iii) the inhibitor is not a phospholipid.

Preferred inhibitors and substantially water-insoluble substances foruse in this embodiment are as hereinbefore defined in relation to thefirst aspect of the present invention.

The invention is further illustrated by the following examples in whichall parts are parts by weight unless stated otherwise.

Particle sizes are quoted as the intensity-averaged particle sizedetermined by dynamic light scattering using a Coulter N4MD.

Pharmacological Properties

The dispersions and particles of the present invention are useful forthe treatment of obesity, psychiatric disorders such as psychoticdisorders, schizophrenia, bipolar disorders, anxiety, anxio-depressivedisorders, depression, cognitive disorders, memory disorders,obsessive-compulsive disorders, anorexia, bulimia, attention disorderslike ADHD, epilepsy, and related conditions, and neurological disorderssuch as dementia, neurological disorders (e.g. Multiple Sclerosis),Raynaud's syndrome, Parkinson's disease, Huntington's chorea andAlzheimer's disease. The dispersions and particles of the presentinvention are also potentially useful for the treatment of immune,cardiovascular, reproductive and endocrine disorders, septic shock anddiseases related to the respiratory and gastrointestinal systems (e.g.diarrhea). The dispersions and particles of the present invention arealso potentially useful as agents in treatment of extended abuse,addiction and/or relapse indications, e.g. treating drug (nicotine,ethanol, cocaine, opiates, etc) dependence and/or treating drug(nicotine, ethanol, cocaine, opiates, etc) withdrawal symptoms. Thedispersions and particles of the present invention may also eliminatethe increase in weight which normally accompanies the cessation ofsmoking.

In a further aspect the present invention provides the use of adispersion of a compound of formula I in the preparation of a medicamentfor the treatment or prophylaxis of obesity, psychiatric disorders suchas psychotic disorders, schizophrenia, bipolar disorders, anxiety,anxio-depressive disorders, depression, cognitive disorders, memorydisorders, obsessive-compulsive disorders, anorexia, bulimia, attentiondisorders like ADHD, epilepsy, and related conditions, neurologicaldisorders such as dementia, neurological disorders (e.g. MultipleSclerosis), Parkinson's Disease, Huntington's Chorea and Alzheimer'sDisease, immune, cardiovascular, reproductive and endocrine disorders,septic shock, diseases related to the respiratory and gastrointestinalsystems (e.g. diarrhea), and extended abuse, addiction and/or relapseindications, e.g. treating drug (nicotine, ethanol, cocaine, opiates,etc) dependence and/or treating drug (nicotine, ethanol, cocaine,opiates, etc) withdrawal symptoms.

In a still further aspect the present invention provides a method oftreating obesity, psychiatric disorders such as psychotic disorders suchas schizophrenia and bipolar disorders, anxiety, anxio-depressivedisorders, depression, cognitive disorders, memory disorders,obsessive-compulsive disorders, anorexia, bulimia, attention disorderslike ADHD, epilepsy, and related conditions, neurological disorders suchas dementia, neurological disorders (e.g. Multiple Sclerosis),Parkinson's Disease, Huntington's Chorea and Alzheimer's Disease,immune, cardiovascular, reproductive and endocrine disorders, septicshock, diseases related to the respiratory and gastrointestinal systems(e.g. diarrhea), and extended abuse, addiction and/or relapseindications, e.g. treating drug (nicotine, ethanol, cocaine, opiates,etc) dependence and/or treating drug (nicotine, ethanol, cocaine,opiates, etc) withdrawal symptoms comprising administering apharmacologically effective amount of a compound of formula I as adispersion and or as particles of the present invention to a patient inneed thereof.

The dispersions and particles of the present invention of the presentinvention are particulary suitable for the treatment of obesity, e.g. byreduction of appetite and body weight, maintenance of weight reductionand prevention of rebound.

EXAMPLE 15,6-bis(4-chlorophenyl)-N-piperidin-1-ylpyrazine-2-carboxamide/Miglyol812 N (4:1 w/w) Dispersion

A solution of 300 mM5,6-bis(4-chlorophenyl)-N-piperidin-1-ylpyrazine-2-carboxamide and 32.1mg/ml Miglyol 812N in dimethylacetamide (DMA) was prepared. 0.15 ml ofthis solution was added rapidly to 2.85 ml of an aqueous solutioncontaining 0.2% w/w polyvinylpyrrolidone (PVP) and 0.25 mM sodiumdodecyl sulfate (SDS). The aqueous solution was sonicated during theaddition of the organic solution using an ultrasonic bath. This resultedin the precipitation of particles with a mean size of 165 nm, asmeasured by dynamic light scattering using a Coulter N4MD. No increasein particle size was observed over a period of 2 hours, at 20° C.

Preparation of Compounds of Formula I

ABBREVIATIONS

-   DCM—dichloromethane-   DMF—dimethylformamide-   DMAP—4-dimethylaminopyridine-   EDC—1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-   TEA—triethylamine-   TFA—trifluoroacetic acid-   DMSO-dimethyl sulfoxide-   DEA—Diethylamine-   PCC—Pyridinium chlorochromate-   DCM—Dichloromethane-   t triplet-   s singlet-   d doublet-   q quartet-   qvint quintet-   m multiplet-   br broad-   bs broad singlet-   dm doublet of multiplet-   bt broad triplet-   dd doublet of doublet    General Experimental Procedures

Mass spectra were recorded on either a Micromass ZQ single quadrupole ora Micromass LCZ single quadrupole mass spectrometer both equipped with apneumatically assisted electrospray interface (LC-MS). ¹H NMRmeasurements were performed on either a Varian Mercury 300 or a VarianInova 500, operating at ¹H frequencies of 300 and 500 MHz respectively.Chemical shifts are given in ppm with CDCl₃ as internal standard.Purification was performed on a semipreparative HPLC with a masstriggered fraction collector, Shimadzu QP 8000 single quadrupole massspectrometer equipped with 19×100 mm C8 column. The mobile phase usedwas, if nothing else is stated, acetonitrile and buffer (0.1 MNH₄Ac:acetonitrile 95:5).

For isolation of isomers, a Kromasil CN E9344 (250×20 mm i.d.) columnwas used. Heptane:ethyl acetate:DEA 95:5:0.1 was used as mobile phase (1ml/min). Fraction collection was guided using a UV-detector (330 nm).

Synthesis of Intermediates

The following intermediates were not commercially available andtherefore prepared as described in Preparation A, (Chem. Ber., 100,1967, p. 555).

Preparation A

(a) 5,6-diphenyl-pyrazine-2-carboxylic acid

The monohydrochloride of 2,3-diaminopropionic acid (500 mg, 3.56 mmol)and benzil (890 mg, 4.23 mmol) were added to a solution of sodiumhydroxide (677 mg, 16.93 mmol) in methanol (10 ml). An extra portion ofmethanol was added (5 ml) and the reaction mixture was refluxed for 20minutes. The mixture was cooled to 25° C. and air was bubbled throughfor 30 minutes. Hydrochloric acid (aq, 2 M) was added until the reactionmixture reached pH 2. The solution was extracted with diethyl ether. Thecombined diethyl ether phases were dried (MgSO₄), filtrated andevaporated under reduced pressure to give the crude product. MS m/z 277(M+H)⁺. The crude product was used in steps described below withoutfurther purification.

(b) 5,6-Bis-(4-bromophenyl)-pyrazine-2-carboxylic acid

The title compound was prepared essentially as described in PreparationA step (a), using monohydrochloride of 2,3-diaminopropionic acid (600mg, 4.26 mmol) and 4,4′-dibromobenzil (1.745 g, 4.26 mmol, 90%) asstarting materials. The reaction mixture was refluxed for 2 hours andair was bubbled through for 1 hour. Hydrochloric acid (aq, 2 M) wasadded until pH 2. The mixture was evaporated under reduced pressure andthe residue was dissolved in water. The solution was extracted withdiethyl ether, the combined diethyl ether phases were dried (MgSO₄),filtered and evaporated under reduced pressure. The crude product (500mg, 27%) was used in steps described below without further purification.MS m/z 435, 437, 439 (M+H)⁺.

(c) 5,6-Di-p-tolyl-pyrazine-2-carboxylic acid

The title compound was prepared as described in Preparation A step (a)using 4,4′-dimethylbenzil (848 mg, 3.56 mmol). The reaction mixture washowever refluxed for 1 hour and air was bubbled through the reactionmixture for about 7 hours. The mixture was evaporated and the residuewas dissolved in water. Hydrochloric acid (aq, 2 M) was added until pH 2was reached. The solution was extracted with diethyl ether. The combineddiethyl ether phases were dried (MgSO₄), filtered and evaporated underreduced pressure. The crude product (918 mg, 85%) was used in stepsdescribed below without further purification. MS m/z 305 (M+H)⁺.

(d) 5,6-Bis-(4-methoxyphenyl)pyrazine-2-carboxylic acid

The title compound was prepared as described in Preparation A step (c)using 4,4′-dimethoxybenzil (961 mg, 3,56 mmol) as starting material. Thereaction mixture was refluxed over night and air was bubbled through themixture for 8 hours. The crude product (435 mg, 36%) was used in stepsdescribed below without further purification. MS m/z 335 (M+H)+

(e) 5,6-Bis-(4-chlorophenyl) pyrazine-2-carboxylic acid

The title compound was prepared as described in Preparation A step (c)using 4,4′-dichlorobenzil (993 mg, 3.56 mmol). Reflux for 1 hour gavedirectly the crude product (923 mg, 75%) that was used in stepsdescribed below without further purification. MS m/z 343, 345, 347(M−H)⁻.

(f) 5,6-Bis-(2-chlorophenyl)pyrazine-2-carboxylic acid

The title compound was prepared as described in Preparation A step (c)using 2,2′-dichlorobenzil (993 mg, 3.56 mmol). The crude product (895mg, 73%) was used in steps described below without further purification.MS m/z 343, 345, 347 (M−H)⁻.

(h) 1-(4-Chlorophenyl)-2-(2,4-dichlorophenyl)ethane-1,2-dione

2-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)ethanone (2.7 g, 9.01 mmol) wasdissolved in 1,2-dichloroethane (25 ml) and freshly made PCC (3.89 g,18.02 mmol), pyridine (1.43 g, 18.02 mmol) and molecular sieves wereadded. The reaction mixture was refluxed under inert atmosphereovernight. The solution was cooled to 25° C., filtered through Silicaand then solvent was evaporated under reduced pressure. The crudeproduct (1.9 g, 66%) was used directly in the next step. ¹H NMR (500MHz) δ 7.97 (d, 2H), 7.84 (d, 1H), 7.52 (d, 2H), 7.46 (s, 1H), 7.44 (d,1H).

(i) 5-(4-Chlorophenyl)-6-(2,4-dichlorophenyl)pyrazine-2-carboxylic acidand 6-(4-chlorophenyl)-5-(2,4-dichlorophenyl)pyrazine-2-carboxylic acid

The title compounds were prepared as described in Preparation A step(a), using 1-(4-chlorophenyl)-2-(2,4-dichlorophenyl)ethane-1,2-dione(1.85 g; 5.90 mmol) from Preparation A step (g) and the monochloride of2,3-diaminopropionic acid (0.61 g, 5.90 mmol) as starting materials. Themixture was refluxed for 30 minutes and then directly worked-up. Thecrude product was allowed to stand over night to aromatise. Flashchromatography (SiO₂, DCM:methanol 10:1, 1% Acetic acid) gave the isomermixture (0.2 g, 10%). MS m/z 377, 379, 381 (M−H)⁻.

EXAMPLE 1 N-(1-piperidinyl)-5,6-diphenyl-2-pyrazinecarboxamide

5,6-Diphenyl-pyrazine-2-carboxylic acid (500 mg, 1.81 mmol) fromPreparation A, step (a), was dissolved in DCM (4 ml) and DMF (150 μl).DMAP (22 mg, 0.18 mmol) and 1-aminopiperidine (218 mg, 2.17 mmol) wereadded and the solution was cooled to 0° C. A slurry of EDC (1.99 mmol,in 2 mL DCM and 100 μl DMF) was added dropwise. The reaction mixture wasstirred at 25° C. After 17 hours additional 1-aminopiperidine (40 mg,0.40 mmol) and EDC (76 mg, 0.40 mmol) was added, and the mixture wasstirred for an additional 3 hours. The crude was diluted with DCM (5 ml)and washed with a saturated solution of NaHCO₃. The organic phase wasdried (MgSO₄), filtered and evaporated. Flash chromatography (SiO₂,ethyl acetate:hexane 2:1) gave the subtitle compound (160 mg, 25%) as awhite solid.

¹H NMR (300 MHz) δ 9.41 (s, 1H), 8.52 (s, 1H), 7.50-7.29 (m, 10H), 2.94(t, 4H), 1.81 (m, 4H), 1.50 (m, 2H).

MS m/z 359 (M+H)⁺.

EXAMPLE 2 N-(1-piperidinyl)-5,6-bis(4-bromophenyl)-2-pyrazinecarboxamide

To 5,6-Bis-(4-bromophenyl)-pyrazine-2-carboxylic acid (108 mg, 0.25mmol) from Preparation A, step (b), DMAP (0.025 mmol, in 500 μl DCM),1-aminopiperidine (0.25 mmol, in 1100 μl DCM), EDC (0.27 mmol, in 1100μl DCM and cooled to 8° C.) were added. The reaction mixture was stirredat 25° C. for 20 h, then washed with saturated NaHCO₃ solution, dried(MgSO₄), filtered and evaporated. Semipreparatory HPLC (0.01% TEA in thebuffered phase) gave the subtitle compound (6.7 mg, 5.4%).

¹H NMR (300 MHz) δ 9.41 (s, 1H), 8.48 (s, 1H), 7.54 (d, 2H), 7.51 (d,2H), 7.36 (d, 2H), 7.34 (d, 2H), 2.94 (t, 4H), 1.81 (m, 4H), 1.55-1.45(m, 2H).

MS m/z 515, 517, 519 (M+H)⁺.

EXAMPLE 3N-(1-piperidinyl)-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide

5,6-Di-p-tolyl-pyrazine-2-carboxylic acid (76 mg, 0.25 mmol) fromPreparation A, step (c), was used as described in Example 2 to give thetitle compound (27 mg, 28%).

¹H NMR (300 MHz) δ 9.35 (s, 1H), 8.57 (s, 1H), 7.38 (d, 4H), 7.18 (d,2H), 7.13 (d, 2H), 2.92 (t, 4H), 2.40 (s, 3H), 2.37 (s, 3H), 1.86-1.75(m, 4H), 1.54-1.44 (m, 2H).

MS m/z 387 (M+H)⁺.

EXAMPLE 4N-(1-piperidinyl)-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide

5,6-Bis-(4-methoxyphenyl)-pyrazine-2-carboxylic acid (84 mg, 0.25 mmol)from Preparation A, step (d), was used as described Example 2 to givethe title compound (20 mg, 19%).

¹H NMR (300 MHz) δ 9.31 (s, 1H), 8.57 (s, 1H), 7.46 (d, 2H), 7.44 (d,2H), 6.90 (d, 2H), 6.86 (d, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 2.93 (t,4H), 1.80 (m, 4H), 1.54-1.45 (m, 2H).

MS m/z 419 (M+H)⁺.

EXAMPLE 5N-(1-piperidinyl)-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide

5,6-Bis-(4-chlorophenyl)-pyrazine-2-carboxylic acid (86 mg, 0.25 mmol)from Preparation A, step (e), was used as described in Example 2 to givethe subtitle compound (16 mg, 15%).

¹H NMR (300 MHz) δ 9.40 (s, 1H), 8.49 (s, 1H), 7.45-7.31 (m, 8H), 2.94(t, 4H), 1.80 (m, 4H), 1.54-1.45 (m, 2H).

MS m/z 427, 429, 431 (M+H)⁺.

EXAMPLE 6N-(1-piperidinyl)-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide

5,6-Bis-(2-chlorophenyl)-pyrazine-2-carboxylic acid (86 mg, 0.25 mmol)from Preparation A, step (f), was used as described in Example 2 to givethe subtitle compound (6 mg, 6%).

¹H NMR (300 MHz) δ 9.52 (s, 1H), 8.52 (s, 1H), 7.44-7.17 (d, 8H),2.94-2.88 (t, 4H), 1.85-1.70 (m, 4H), 1.52-1.44 (m, 2H).

MS m/z 427, 429, 431 (M+H)⁺.

EXAMPLE 7 N-cyclohexyl-5,6-diphenyl-2-pyrazinecarboxamide

5,6-diphenyl-pyrazine-2-carboxylic acid (70 mg, 0.25 mmol) fromPreparation A, step (a), was reacted essentially as described in Example2 but with cyclohexylamine (0.25 mmol, in 1 ml DCM), DMAP (0.025 mmol,in 0.5 ml DCM), EDC (0.28 mmol, in 1 ml DCM, and cooled to 8° C.) andDMF (100 μl). Semipreparatory HPLC (0.15% TFA/water:acetonitrile 95:5instead of the buffer phase) gave the title compound (7 mg, 8%) afterwashing with Na₂CO₃ solution.

¹H NMR (300 MHz) δ 9.41 (s, 1H), 7.78 (d, 1H), 7.49-7.28 (m, 10H),4.12-3.97 (m, 1H), 2.13-1.23 (m, 10H).

MS m/z 358 (M+H)⁺.

EXAMPLE 8 N-cyclohexyl-5,6-bis(4-bromophenyl)-2-pyrazinecarboxamide

5,6-Bis-(4-bromophenyl)-pyrazine-2-carboxylic acid (109 mg, 0.25 mmol)from Preparation A, step (b), was used as described in Example 7.Semipreparatory HPLC (0.15% TFA/water:acetonitrile 95:5 instead of thebuffer phase) gave the title compound (7 mg, 8%) after washing withNa₂CO₃ solution.

¹H NMR (300 MHz) δ 9.41 (s, 1H), 7.68 (s, 1H), 7.54 (d, 2H), 7.50 (d,2H), 7.36 (d, 2H), 7.34 (d, 2H), 4.11-3.96(m, 1H), 2.12-1.20 (m, 10H).

MS m/z 514, 516, 518 (M+H)⁺.

EXAMPLE 9 N-cyclohexyl-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide

5,6-Di-p-tolyl-pyrazine-2-carboxylic acid (76 mg, 0.25 mmol) fromPreparation A, step (c), was used as described in Example 7.Semipreparatory HPLC (0.01% TEA in the buffer phase) gave the subtitlecompound (4 mg, 4%).

¹H NMR (300 MHz) δ 9.36 (s, 1H), 7.77 (d, 1H), 7.39 (d, 4H), 7.18 (d,2H), 7.13 (d, 2H), 4.10-3.96 (m, 1H), 2.40 (s, 3H), 2.37 (s, 3H),2.09-1.20 (m, 10H).

MS m/z 386 (M+H)⁺.

EXAMPLE 10 N-cyclohexyl-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide

5,6-Bis-(4-methoxyphenyl)-pyrazine-2-carboxylic acid (76 mg, 0.25 mmol)from Preparation A, step (d), was used essentially as described inExample 7 but the reaction mixture was first stirred overnight, thenmore cyclohexylamine (25 mg, 0.25 mmol) was added and the mixture wasstirred for an additional two days prior to workup. Semipreparatory HPLC(0.15% TFA in the buffered phase) gave the title compound (12 mg, 11%).

¹H NMR (300 MHz) δ 9.32 (s, 1H), 7.76 (d, 1H), 7.47 (d, 2H), 7.45 (d,2H), 6.90 (d, 2H), 6.86 (d, 2H), 4.10-3.96 (m, 1H), 3.86 (s, 3H), 3.84(s, 3H), 2.09-1.17 (m, 10H).

MS m/z 418 (M+H)⁺.

EXAMPLE 11 N-cyclohexyl-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide

5,6-Bis-(4-chlorophenyl)pyrazine-2-carboxylic acid (86 mg, 0.25 mmol)from Preparation A, step (e), was used as described in Example 10 togive the title compound (7 mg, 8%) after washing with Na₂CO₃ solution.

¹H NMR (300 MHz) δ 9.41 (s, 1H), 7.69 (s, 1H), 7.47-7.30 (m, 8H),4.10-3.97 (m, 1H), 2.10-1.18 (m, 10H).

MS m/z 426, 428, 430 (M+H)⁺.

EXAMPLE 12 N-cyclohexyl-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide

5,6-Bis-(2-chlorophenyl)-pyrazine-2-carboxylic acid (86 mg, 0.25 mmol)from preparation A step (f) was used as described in Example 10, to givethe title compound (14 mg, 13%).

¹H NMR (300 MHz) δ 9.51 (s, 1H), 7.74 (s, 1H), 7.41-7.18 (m, 8H),4.10-3.97 (m, 1H), 2.07-1.14 (m, 10H).

MS m/z 426, 428, 430 (M+H)⁺.

EXAMPLE 13 N,5,6-triphenyl-2-pyrazinecarboxamide

To 5,6-Diphenyl-pyrazine-2-carboxylic acid (70 mg, 0.25 mmol) fromPreparation A, step (a), DMAP (0.025 mmol, in 0.5 ml DCM), aniline (0.25mmol, in 1 ml DCM), EDC (0.28 mmol, in 1 ml DCM, cooled to 8° C.) andDMF (100 μl) were added. The reaction mixture was stirred at 25° C. overnight, then worked up as described in Example 2. Semipreparatory HPLC(0.15% TFA/water:acetonitrile 95:5 instead of the buffer phase) gave thetitle compound (27 mg, 30%) after washing with Na₂CO₃ solution.

¹H NMR (300 MHz) δ 9.75 (s, 1H), 9.52 (d, 1H), 7.80 (d, 2H), 7.55-7.32(m, 12H), 7.20 (t, 1H).

MS m/z 352 (M+H)⁺.

EXAMPLE 14 N-phenyl-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide

5,6-Di-p-tolyl-pyrazine-2-carboxylic acid (77 mg, 0.25 mmol) fromPreparation A, step (c), was used as described in Example 13 to give thesubtitle compound (28 mg, 29%).

¹H NMR (500 MHz) δ 9.78 (s, 1H), 9.49 (s, 1H), 7.81 (d, 2H), 7.47-7.43(m, 6H), 7.25-7.17 (m, 5H), 2.45 (s, 3H), 2.41 (s, 3H).

MS m/z 380 (M+H)⁺.

EXAMPLE 15 N-phenyl-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide

5,6-Bis-(4-methoxyphenyl)-pyrazine-2-carboxylic acid (85 mg, 0.25 mmol)from Preparation A step (d), was used as described in Example 13, togive the title compound (33 mg, 32%).

¹H NMR (300 MHz) δ 9.74 (s, 1H), 9.42 (s, 1H), 7.79 (d, 2H), 7.50 (d,4H), 7.42 (t, 2H), 7.19 (t, 1H), 6.94 (d, 2H), 6.89 (d, 2H), 3.88 (s,3H), 3.85 (s, 3H).

MS m/z 412 (M+H)⁺.

EXAMPLE 16 N-phenyl-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide

5,6-Bis-(4-chlorophenyl)-pyrazine-2-carboxylic acid (87 mg, 0.25 mmol)from Preparation A, step (e), was used as described in Example 13, togive the subtitle compound (6 mg, 6%).

¹H NMR (300 MHz) δ 9.66 (s, 1H), 9.52 (s, 1H), 7.79 (d, 2H), 7.48-7.35(m, 10H), 7.21 (t, 1H).

MS m/z 420, 422, 424 (+H)⁺.

EXAMPLE 17 N-phenyl-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide

5,6-Bis-(2-chloro-phenyl)-pyrazine-2-carboxylic acid (87 mg, 0.25 mmol)from Preparation A, step (f), was treated as described in Example 13, togive the title compound (27 mg, 25%).

¹H NMR (500 MHz) δ 9.73 (s, 1H), 9.66 (s, 1H), 7.81 (d, 2H), 7.46-7.22(m, 11H).

MS m/z 420, 422, 424 (M+H)⁺.

EXAMPLE 185-(4-Chlorophenyl)-6-(2,4-dichlorophenyl)pyrazine-2-carboxylic acidpiperidin-1-ylamide and6-(4-chlorophenyl)-5-(2,4-dichlorophenyl)pyrazine-2-carboxylic acidpiperidin-1-ylamide

The mixture of5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)pyrazine-2-carboxylic acid and6-(4-chlorophenyl)-5-(2,4-dichlorophenyl)pyrazine-2-carboxylic acid (78mg, 0.205 mmol) from Preparation A step (i) and thionyl chloride (147mg, 1.23 mmol) were refluxed in toluene (2 ml) for 3 hours. The solventand reagents were evaporated under reduced pressure and theintermediates were dissolved in DCM (1 ml). TEA (42 mg, 0.41 mmol) and1-aminopiperidine (21 mg, 0.205 mmol) were dissolved in DCM (1 ml) andadded. The reaction mixture was stirred at 25° C. overnight and thenevaporated under reduced pressure. Flash chromatography (SiO₂,heptane:ethyl acetate 1:1) gave a mixture of the title compounds (45 mg,47%, ratio of isomers 0.5:1). ¹H NMR (300 MHz) δ 9.46 (s, 1H), 8.39 (s,1H), 7.47-7.28 (m, 7H), 3.02-2.84 (m, 4H), 1.89-1.73 (m, 4H), 1.57-1.41(m, 2H) and 9.42 (s, 1H), 8.51 (s, 1H), 7.47-7.28 (m, 7H), 3.02-2.84 (m,4H), 1.89-1.73 (m, 4H), 1.57-1.41 (m, 2H).

EXAMPLE 18 (A)N-(1-piperidinyl)-5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)-2-pyrazinecarboxamide

The title compound was isolated from the mixture prepared in Example 18(35 mg) by preparative chromatography (9 mg, 26%). ¹H NMR (300 MHz) δ9.46 (s, 1H), 8.38 (s, 1H), 7.46-7.24 (m, 7H), 2.89 (t, 4H), 1.78 (p,4H), 1.52-1.40 (m, 2H).

EXAMPLE 18 (B)N-(1-piperidinyl)-6-(4-chlorophenyl)-5-(2,4-dichlorophenyl)-2-pyrazinecarboxamide

The title compound was isolated from the mixture prepared in Example 18(35 mg) by preparative chromatography (11 mg, 31%). ¹H NMR (300 MHz) δ9.42 (s, 1H), 8.50 (s, 1H), 7.39-7.30 (m, 7H), 2.93 (t, 4H), 1.80 (p,4H), 1.54-1.43 (m, 2H).

Pharmacological Activity

Compounds of Formula I are active against the receptor product of theCB1 gene. The affinity of the compounds of the invention for centralcannabinoid receptors is demonstrable in methods described in Devane etal, Molecular Pharmacology, 1988, 34,605 or those described inWO01/70700 or EP 656354. Alternatively the assay may be performed asfollows.

10 μg of membranes prepared from cells stably transfected with the CB1gene were suspended in 200 μl of 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 5,mM HEPES (pH 7.4), 1 mM DTT, 0.1% BSA and 100 μM GDP. To this was addedan EC80 concentration of agonist (CP55940), the required concentrationof test compound and 0.1 μCi [³⁵S]-GTPγS. The reaction was allowed toproceed at 30° C. for 45 min. Samples were then transferred on to GF/Bfilters using a cell harvester and washed with wash buffer (50 mM Tris(pH 7.4), 5 mM MgCl₂, 50 mM NaCl). Filters were then covered withscintilant and counted for the amount of [³⁵S]-GTPγS retained by thefilter.

Activity is measured in the absence of all ligands (minimum activity) orin the presence of an EC80 concentration of CP55940 (maximum activity).These activities are set as 0% and 100% activity respectively. Atvarious concentrations of novel ligand, activity is calculated as apercentage of the maximum activity and plotted. The data are fittedusing the equation y=A+((B−A)/1+((C/x)UD)) and the IC50 value determinedas the concentration required to give half maximal inhibition of GTPγSbinding under the conditions used.

The compounds of Formula I are active at the CB1 receptor (IC50<1micromolar). Most preferred compounds have IC50<200 nanomolar.

1. A process for the preparation of a stable dispersion of solidparticles in an aqueous medium comprising: combining (a) a firstsolution comprising a substantially water-insoluble substance which is acompound of Formula I

or a pharmaceutically acceptable salt thereof, in which R¹ and R²independently are selected from a C₁₋₆alkyl group; an (amino)C₁₋₄alkyl-group in which the amino is optionally substituted by one or moreC₁₋₃alkyl groups; an optionally substituted non-aromaticC₃₋₁₅carbocyclic group; a (C₃₋₁₂cycloalkyl)C₁₋₃alkyl- group; agroup—(CH₂)_(r)(phenyl)_(s) in which r is 0, 1, 2, 3 or 4, s is 1 when ris 0 otherwise s is 1 or 2 and the phenyl groups are optionallyindependently substituted by one, two or three groups represented by Z;naphthyl; anthracenyl; a saturated 5 to 8 membered heterocyclic groupcontaining one nitrogen and optionally one of the following: oxygen,sulphur or an additional nitrogen wherein the heterocyclic group isoptionally substituted by one or more C₁₋₃alkyl groups, hydroxy orbenzyl; 1-adamantylmethyl; and a group —(CH₂)_(t) Het in which t is 0,1, 2, 3 or 4, and the —(CH₂)_(t) moiety is optionally substituted by oneor more C₁₋₃alkyl groups and Het represents an aromatic heterocycleoptionally substituted by one, two or three groups selected from aC₁₋₅alkyl group, a C₁₋₅alkoxy group and halo; or R¹ represents H and R²is as defined above; or R¹ and R² together with the nitrogen atom towhich they are attached represent a saturated 5 to 8 memberedheterocyclic group containing one nitrogen and optionally one of thefollowing: oxygen, sulphur or an additional nitrogen; wherein theheterocyclic group is optionally substituted by one or more C₁₋₃alkylgroups, hydroxy or benzyl; X is CO or SO₂; Y is absent or is NHoptionally substitututed by a C₁₋₃alkyl group; R³ and R⁴ independentlyare selected from phenyl, thienyl and pyridyl, each of which isoptionally substituted by one, two or three groups represented by Z; Zrepresents a C₁₋₃alkyl group, a C₁₋₃alkoxy group, hydroxy, halo,trifluoromethyl, trifluoromethylthio, trifluorornethoxy,trifluoromethylsulphonyl, nitro, amino, mono or di C₁₋₃alkylamino, monoor di C₁₋₃alkylamido, C₁₋₃alkylsulphonyl, C₁₋₃alkoxycarbonyl, carboxy,cyano, carbamoyl, mono or di C₁₋₃alkyl carbamoyl, sulphamoyl or acetyl;and R⁵ is H, a C₁₋₃alkyl group, a C₁₋₃alkoxymethyl group,trifluoromethyl, a hydroxyC₁₋₃alkyl group, C₁₋₃alkoxycarbonyl, carboxy,cyano, carbamoyl, mono or di C₁₋₃alkylcarbamoyl, acetyl, orhydrazinocarbonyl of formula —CONHNR^(a)R^(b) wherein R^(a) and R^(b)are as previously defined for R¹ and R² respectively; a water-miscibleorganic solvent and an inhibitor with (b) an aqueous phase comprisingwater and optionally a stabiliser, thereby precipitating solid particlescomprising the inhibitor and the substantially water-insolublesubstance; and optionally removing the water-miscible organic solvent;wherein: the inhibitor is less soluble in water than the water-insolublesubstance, and the inhibitor is selected from one or more of: (i) amono-, di- or a tri-glyceride of a fatty acid; (ii) a fatty acid mono-or di-ester of a C₂₋₁₀ diol; (iii) a fatty acid ester of an alkanol or acycloalkanol; (iv) a wax; (v) a long chain aliphatic alcohol of 6 to 30carbon atoms; and (vi) a hydrogenated vegetable oil.
 2. A processaccording to claim 1, wherein the inhibitor is a mixture oftriglycerides obtainable by esterifying glycerol with a mixture ofmedium chain fatty acids.
 3. A process according to claim 2, wherein theinhibitor is a mixture of triglycerides containing acyl groups with from8 to 12 carbon atoms.
 4. A process according to claim 1, wherein theinhibitor further comprises a co-inhibitor selected from a long chainaliphatic alcohol containing 6 30 carbon atoms.
 5. A process accordingto claim 1, wherein the inhibitor is sufficiently miscible with thesubstantially water-insoluble substance to form solid particles in thedispersion comprising a substantially single phase mixture of thesubstance and the inhibitor.
 6. A process according to claim 1, whereinthe miscibility of the inhibitor and substantially water-insolublesubstance is sufficient to give an interaction parameter χ of less than2.5.
 7. A process according to claim 1, wherein the aqueous phasecontains a stabiliser.
 8. A process according to claim 7, wherein thestabiliser comprises a polymeric dispersant and a surfactant.
 9. Aprocess according to claim 1, wherein the mean particle size of thesolid particles is less than 1 μm.
 10. A process according to claim 1,farther comprising isolating the solid particles from the dispersion.11. A process for the preparation of a stable dispersion of solidparticles in an aqueous medium comprising combining (a) a first solutioncomprising a substantially water-insoluble substance, a water-miscibleorganic solvent, and an inhibitor with (b) an aqueous phase comprisingwater and optionally, a stabilizer, thereby precipitating solidparticles comprising the inhibitor and the substantially water-insolublesubstance; and optionally removing the water-miscible organic solvent;wherein: the inhibitor is less soluble in water than the water-insolublesubstance, and the inhibitor is selected from one or more of: a mono-,di- or a tri-glyceride of a fatty acid; (ii) a fatty acid mono- ordi-ester of a C₂₋₁₀ diol; (iii) a fatty acid ester of an alkanol or acycloalkanol; (iv) a wax; (v) a long chain aliphatic alcohol of 6 to 30carbon atoms; and (vi) a hydrogenated vegetable oil; and wherein thesubstantially water-insoluble substance is selected from:N-(1-piperidinyl)-5,6-diphenyl-2-pyrazinecarboxamide;N-(1-piperidinyl)-5,6-bis(4-bromophenyl)-2pyrazinecarboxamide;N-(1-piperidinyl)-5,6-bis(4-bromophenyl)-2pyrazinecarboxamide;N-(1-piperidinyl)-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide;N-(1-piperidinyl)-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide;N-(1-piperidinyl)-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide;N-cyclohexyl-5,6-diphenyl-2-pyrazinecarboxamide;N-cyclohexyl-5,6-bis(4-bromophenyl)-2-pyrazinecarboxamide;N-cyclohexyl-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide;N-cyclohexyl-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide;N-cyclohexyl-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide;N-cyclohexyl-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide;N,5,6-triphenyl-2-pyrazinecarboxamide;N-phenyl-5,6-bis(4-methylphenyl)-2-pyrazinecarboxamide;N-phenyl-5,6-bis(4-methoxyphenyl)-2-pyrazinecarboxamide;N-phenyl-5,6-bis(4-chlorophenyl)-2-pyrazinecarboxamide;N-phenyl-5,6-bis(2-chlorophenyl)-2-pyrazinecarboxamide;N-(1-piperidinyl)-5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)-2-pyrazinecarboxamide;andN-(1-piperidinyl)-6-(4-chlorophenyl)-5-(2,4-dichlorophenyl)-2-pyrazinecarboxamide;or, where applicable, an optical isomer, tautomer, stereoisomer, orracemate thereof, or a pharmaceutically acceptable salt thereof.